1a.Objectives (from AD-416):
The overall objective of this project is to develop commercially viable methods for the control of pathogens in tree nuts (almonds, pistachios and walnuts) and raisins which lead to foodborne illness in humans and animals. This includes both the control of fungal pathogens (mycotoxins) as well as bacterial pathogens such as E. coli and Salmonella. One approach will be the development of biocontrol agents using bacteria, yeast and non-toxigenic Aspergillus carbonarius. In addition, a better understanding of organic and conventional farming systems will provide new insights on mycotoxin control. The specific objectives for the period covered by this project plan are as follows:
Objective 1: Define the critical control points for pathogen contamination during the production stream. Place particular emphasis on agricultural water sources including dairy waste water. Using both cultural and non-cultural based methods we will identify the points in the developmental process, as well as the processing cycle where tree nuts are most likely to become contaminated with human pathogenic bacteria and mycotoxigenic fungi.
Objective 2: Evaluate the microbial ecology of organic v. conventional practices. Although several surveys have reported that consumers equate organically grown food stuffs with higher levels of food safety, little is known about how these practices affect the microbial population structure or mycotoxin levels in tree nuts. We will address the influence of phyllosphere microbial community on the population diversity of A. flavus in tree nut orchards, and A. carbonarius on grape surfaces in both farming systems.
Objective 3: Delineate the factors affecting cross-contamination during processing and develop a potential intervention strategy during storage. A number of experiments point to a strong possibility that cross-contamination of toxigenic fungi is possible during processing and storage, although no research appears to have been done on the transfer in actual (not laboratory) processing and storage conditions. Development of novel approaches to prevent the growth of storage fungi and production of harmful toxins is a high priority in the almond industry. Edible films and coatings (EFC) containing antimicrobial natural compounds will be tested for their efficiency to reduce mycotoxin in stored almonds.
Objective 4: Develop biological-control/intervention technologies using competitive or antagonistic microorganisms such as yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post-harvest environments. Bacterial and yeast biocontrol agents will be tested in almond orchards and vineyards to control A. flavus and A. carbonarius, as well as human pathogenic bacteria, E. coli and Salmonella. EPA registration of the patented yeast, Pichia anomala will be pursued for commercial application. Methods to enhance the biocontrol efficacy will be developed.

1b.Approach (from AD-416):
Develop mentods to control insect pests and toxic fungi of tree nuts. Insects include naval orangeworm, codling moth and peach twig borer. Feeding damage by these insects leads to infection by aflatoxigenic aspergilli. Control methods for insects are to be environmentally benign and employ semiochemicals to disrupt insect behavior. Control of toxic fungi focuses on biological control using competitive or antagonistic microorganisms. These microorganisms include either yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post harvest environments.

DNA was extracted from almond surfaces obtained from farms that used both conventional and organic practices and the microbial population structure on them determined using 16S rRNA DNA sequence analysis. Significant differences were at different stages of almond development. Sequences associated with Salmonella enterica were observed in fully mature almonds obtained from one of 3 farms using conventional growing methodologies; while none were obtained from those using organic practices.

Species-specific PCR primer sets for detection and differentiation of four Aspergillus species (based on DNA sequence differences in the calmodulin gene) were developed. These primers have been used to detect these species in soil inoculated with mixtures of Aspergillus species. This analysis will allow faster throughput of field samples to detect the presence of ochratoxin- and fumonisin-producing species.

We have initiated an analysis of Aspergillus population diversity in raisin vineyards beginning at flowering and fruit set, continuing through fruit development and ripening, and ultimately to grape harvesting and drying. Samples are being analyzed for the presence and distribution of mycotoxin-producing Aspergillus species, to determine points at which interventions may be useful for mycotoxin control.

This project investigates aflatoxin cross-contamination from nut to nut during processing of shelled almonds and potential transfer of aflatoxin to processing equipment. The results indicated an approximate 16% transfer of aflatoxin to the Clean almonds. Amount of aflatoxin correlated strongly with the number of Hot nuts in each sample, as expected. It was found that very little aflatoxin transferred to the equipment. Further analysis is pending and a manuscript is under preparation.

Mini-storage bins (250 ml glass jar with caps) were filled with 50 g of raw almonds. Sterile distilled water was added to adjust the water activity of almonds to 0.83. Individual jar was lined with an edible film on the bottom and another film glued to the inside of the lid. Visual observation on fungal growth was conducted monthly. Tomato edible film was found to be effective in preventing fungal growth on almonds.

Fungal cell walls were prepared and added to growth medium. Growth of Pichia anomala cells was monitored. The colony forming units (CFU) were determined when yeast cells reached to stationary phase. Chitosan solution was prepared and its effect on aflatoxin production by Aspergillus flavus was tested. These biopolymers may be useful to increase the yield of biocontrol yeast cells and to inhibit aflatoxin production.

4.Accomplishments
1.
Ochratoxin and fumonisin contamination in almonds. Rejected almond samples were analyzed to determine whether indigenous black-spored Aspergillus species could contaminate almonds with mycotoxins. Isolated species included atoxigenic strains of A. tubingensis and toxigenic strains of A. niger, and A. awamori, which accounted for 72% of fumonisin production. No ochratoxin-producing fungi were isolated. In contrast, almonds contained no detectable fumonisin, but 3 of 21 samples contained low levels of ochratoxin. These results indicate that properly dried and stored almonds are not conducive to fumonisin contamination, and that ochratoxin contamination is sporadic, and possibly caused by other Aspergillus species. This analysis points out future research focus for solving ochratoxin contamination in almonds.

2.
Effect of biopolymer on yeast cells growth and inhibition of aflatoxin biosynthesis. Mass production of biocontrol yeast cells is essential. Fungal cell walls were prepared and added to growth medium. Yeast cells grown in the supplemented medium were stimulated and colony forming units (CFU) were increased by 40%. The biopolymer chitosan was found to be effective in inhibit aflatoxin biosynthesis of Aspergillus flavus by 43% at concentration of 0.05%. Chitosan is a GRAS (generally regarded as safe) biopolymer and can be used in conjuction with biocontrol yeast. The findings can be applied to solve problems of aflatoxin contamination in the almond and corn industries.

3.
Development of commercial products for control of A. flavus and aflatoxin. The patented yeast, Pichia anomala WRL076 has been licensed by Verdesian Life Sciences LLC as a biological control agent antagonistic to mycotoxigenic fungi. Biological control provides a viable alternative to the use of fungicides in agriculture. Moreover, to reduce harmful fungi while having a minimal adverse impact on the environment. Commercial products of Pichia anomala are needed for the control of toxigenic aspergilli and eliminate Mycotoxin contamination on food.